hnRNP A1 KNOCKOUT ANIMAL MODEL AND USE THEREOF

ABSTRACT

A nucleic acid construct comprising a genetic engineered heterogeneous nuclear ribonucleoprotein (hnRNP) A1 gene is provided. A transgenic mouse in which the expression of hnRNP A1 gene has been disrupted is also provided. The mouse is useful for studying the role of hnRNP A1 gene in normal and disease states of a neurodegenerative disease or a cancer for developing therapies to treat any of these diseases. Therefore, a method of screening a compound for potential use in prevention and/or treatment of neurodegenerative disease or cancer is further provided.

BACKGROUND

1. Technical Field

The present disclosure relates to vectors comprising Heterogeneousnuclear ribonucleoprotein (hnRNP) A1 gene and non-human animals in whichthe expression of hnRNP A1 gene has been disrupted.

2. Description of Related Art

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a protein that hasbeen reported to play a significant part in regulating the process ofgene splicing (Del Gatto-Konczak et al., 1999 MOI Cell Biol 19,251-260), telomere extension (LaBranche et al., 1998 Nat Genet 19,199-202), and viral replication (Lin et al., 2009 J Virol 83, 6106-6114;Monette et al., 2009, J Biol Chem 284, 31350-31362), and any of theidentified cellular events has been implicated with diseases includingcancerous progression carcinogenesis, and neurodegenerative disease.

Recently, hnRNP A1 is identified to be involved in alternative splicingof many disease-related proteins, such as GTPase Rac1 andcarcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1).Rac1b, an alternatively spliced isoform of Rac1, was originallyidentified as an over-expressed protein in breast and colorectal cancercells, and has subsequently been suggested an important role in manyoncogenic signaling pathways. CEACAM1 is expressed in a variety of celltypes, including breast cancer cells, and is also implicated incarcinogenesis. Alternative splicing of Exon 11 of the insulin receptorgene (INSR), which is developmental stage-dependent and tissue-specific,is also regulated by hnRNP A1. hnRNP A1 inhibites exon 11 inclusion andresults in insulin receptor-B (IR-B) expression, which predominantlyexpress in insulin-sensitive tissue, suggesting a metabolic role(Talukdar et al., PLoS One (2011), 6: e27869). It is demonstrated thathnRNP A1 is a negative factor to splicing selection of AtaxiaTeleangectasia Mutated gene (ATM), the gene mutated in an autosomalrecessive disorder characterized by cerebellar ataxia and oculocutaneoustelangiectasias (Pastor et al., PLoS One (2011), 6: e23349).

In view of the role of hnRNP A1 associated with various diseases, it isuseful to provide an animal model, particularly, a hnRNP A1 knockoutmodel, in which the expression level of hnRNP A1 protein is notexpressed in null mice and the expression level of hnRNP A1 in theheterozygous in the knockout model is relatively low, as compared to anormal animal, for further studies on the function of hnRNP A1 gene inany of the identified diseases and its use in developing therapies totreat any of these diseases.

SUMMARY

As embodied and broadly described herein, disclosure herein featuresvectors comprising hnRNP A1 gene and non-human animals and cell lines inwhich the expression of hnRNP A1 gene has been disrupted.

Accordingly, in one aspect, the present disclosure is directed to atargeting vector or a nucleic acid construct, which includes a nucleicacid sequence, in which a first locus of recombination sequence 1 isinserted before the exon 2 of the endogeneous hnRNP A1 gene, and secondrecombination sequences 2 flanking a marker gene followed by a secondlocus of recombination sequence 1 is inserted behind the exon 8 of theendogenous hnRNP A1 gene.

In another aspect, the present disclosure is directed to a cellcontaining the nucleic acid construct of the present invention or adisrupted hnRNP A1 gene. Preferably, the cell is a stem cell, and morepreferably, an embryonic stem (ES) cell, and most preferably, a murineES cell. According to one embodiment, the cell is produced byintroducing the nucleic acid construct of the present invention into astem cell to produce a homologous recombinant, resulting in a disruptionin the hnRNP A1 gene, in which the neomycin resistant gene and the exons2 to 8 of the hnRNP A1 gene are respectively deleted by the introductionof FLP recombinase and Cre recombinase.

In still another aspect, the present disclosure provides a non-humananimal and its progeny having a disruption in hnRNP A1 gene. In oneembodiment, the non-human animal and its progeny are heterozygous orhomozygous for a null mutation in the hnRNP A1 gene. In anotherembodiment, the non-human animal and its progeny having a disruption inhnRNP A1 gene exhibit decreased expressed levels of the hnRNP A1 gene,relative to the wild-type non-human animals. Preferably, the non-humananimal and its progeny are rodents and, most preferably, are mice.

In a further aspect, the present disclosure provides a method ofobtaining a non-human animal deficient in hnRNP A1 gene, or withdecreased or null expressed level of hnRNP A1 gene. The method includessteps of inserting into the genome of the embryonic stem cell derivedfrom the non-human animal the nucleic acid construct of the presentinvention, injecting the embryonic stem cell into a blastocyst of thenon-human animal after introduction of appropriate recombinase, andimplanting the blastocyst into the uterus of a foster mother.Preferably, the non-human animal is rodent and, most preferably, ismouse.

The non-human animals of the present disclosure are useful for studyinghnRNP A1 gene and diseases wherein hnRNP A1 gene is implicated,including neurodegenerative disease and cancer. The non-human animals ofthe present disclosure are useful for identifying therapeutic compoundsthat may be useful in preventing and/or treating any of these diseases.

Accordingly, in still a further aspect, the present disclosure providesa method of screening a compound for potential use in modulating thefunction of the hnRNP A1 gene that linked to the prevention and/ortreatment of neurodegenerative disease or cancer. The method includessteps of respectively administering a test compound to the non-humananimal deficient in hnRNP A1 gene and a wild-type non-human animal orcells or tissues derived therefrom; and assessing the function of exonicRNA splicing in each of the non-human animal, cells, or tissues definedabove, prior to and after a given time period of the administration; andcomparing the non-human animal deficient in hnRNP A1 gene with thewild-type non-human animal in terms of the test results to determineeffectiveness of the test compound. Preferably, the non-human animal isrodent and, most preferably, is mouse.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of thepresent invention will be apparent from the description, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings.

FIG. 1 is a schematic drawing illustrating the construction strategy ofa nucleic acid construct having DNA fragments with exons 2 to 8 of hnRNPA1 being deleted for generating hnRNP A1 knockout mice in accordancewith one embodiment of this invention.

FIG. 2A illustrates the genomic PCR analysis of homozygous (−/−),heterozygous (+/−) and wild-type (+/+) alleles of the F1 mice inaccordance with one embodiment of this invention.

FIG. 2B illustrates the expression of hnRNP A1 protein in heart (H) andbrain (B) tissues of the F1 mice in accordance with one embodiment ofthis invention.

FIG. 3A is a picture showing the external morphology of wild-type (+/+),heterozygous (+/−), and homozygous (−/−) hnRNP A1 knockout embryo at dayE18.5 in accordance with one embodiment of this invention.

FIGS. 3B to 3D are pictures showing the sagittal section of wild-type(+/+), heterozygous (+/−), and homozygous (−/−) hnRNP A1 knockout embryoat day E18.5 in accordance with one embodiment of this invention.

FIG. 4 are histoimmuno-staining photographs illustrating the homozygous(−/−) hnRNP A1 knockout mice have diaphragm and urinary bladder defectsin accordance with one embodiment of this invention, in which HEstaining was performed on the diaphragm and urinary bladder ofhomozygous (−/−) hnRNP A1 knockout mouse and control mice, and (A) isthe diaphragm of normal mouse at E18.5, 200×; (B) is the diaphragm ofhomozygous (−/−) hnRNP A1 knockout mouse that showed sarcoplasmicdegeneration and fibrous tissues infiltration at E18.5, 200×; (C) is theurinary bladder of normal mouse at E18.5, 400×; and (D) is the urinarybladder of homozygous (−/−) hnRNP A1 knockout mouse that showedhyperplasia of transitional cells and appearance of several degenerativecells at E18.5, 400×. “m” represents mitosis, and asterisk representsdegenerative cell.

FIG. 5 illustrates the amount of hnRNP A1 protein expressed in organs ofwild-type (+/+) mouse and heterozygous (+/−) hnRNP A1 knockout adultmouse in accordance with one embodiment of this invention, in which thehnRNP A1 protein from lung, testis and brain of wild-type (+/+) mouseand heterozygous (+/−) hnRNP A1 knockout adult mouse was detected byWestern blot and beta-actin was used as loading control.

DETAILED DESCRIPTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example isconstructed or utilized. The description sets forth the functions of theexamples and the sequence of steps for constructing and operating theexamples. However, the same or equivalent functions and sequences may beaccomplished by different examples.

1. Definitions

The terms “a”, “an”, and “the” as used herein are defined to mean “oneor more” and include plural referents unless the context clearlydictates otherwise.

The term “gene” refers to a gene containing at least one of the DNAsequence disclosed herein, or any DNA sequence encodes the amino acidsequence encoded by the DNA sequence disclosed herein, or any DNAsequence that hybridizes to the complement of the coding sequencedisclosed herein. Preferably, the term includes coding and non-codingregions, and preferably all sequences necessary for normal geneexpression including promoters, enhancers, and other regulatorysequences.

The term “nucleic acid” refers to polymeric forms of nucleotides of anylength. The nucleic acid may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimension structure, and may perform any function, known orunknown. The term “nucleic acid” includes single-, double-stranded andtriple helical molecules. Non-limited examples of nucleic acids include,but are not limited to, a gene or its fragment, exons, introns, mRNA,tRNA, rRNA, ribozymes, cDNA, recombinant nucleic acid, plasmids,vectors, isolated DNA or RNA of any sequence, nucleic acid probes andprimers. A nucleic acid may also comprise modified nucleic acidmolecules, such as methylated nucleic acid molecules and nucleic acidanalogs.

The term “homologous recombination” refers to exchange DNA fragmentsbetween two DNA molecules at the site of homologous nucleotide sequence.

The term “homologous” are used herein to denote a characteristic of aDNA sequence having at least 70% sequence identity as compared to areference sequence, typically at least about 85% sequence identity,preferably at least about 95% sequence identity, and more preferablyabout 98% sequence identity, and most preferably about 100% sequenceidentity as compared to a reference sequence. Homology can be determinedusing, for example, a “BLASTN” algorithm. It is understood thathomologous sequence may accommodate insertions, deletions, andsubstitutions in the nucleotide sequence. Thus, linear sequence ofnucleotides can be essentially identical even if some of the nucleotidesresidues do not precisely correspond or align. The reference sequencemay be a subset of a large sequence, such as a portion of a gene orflanking sequence.

“Disruption” of an hnRNP A1 gene occurs when a fragment of genomic DNAlocates and recombines with an endogenous homologous sequence. Thesesequence disruptions or modifications may include insertions, missense,framshift, deletion, substitutions, or replacement of DNA sequence, orany combinations thereof. Insertions include the insertion of the entiregenes, which may be of any origin. Disruption, for example, can alter orreplace a promoter, an enhancer or splice site of the hnRNP A1 gene, andcan alter the normal gene product by inhibiting its productionpartially, or completely. In a preferred embodiment, the disruption is anull disruption, wherein there is no significant expression of the hnRNPA1 gene.

The practices of this invention are hereinafter described in detail withrespect to targeting vectors or nucleic acid constructs comprising anucleic acid sequence comprising hnRNP A1 gene; and non-human animals inwhich the expression of hnRNP A1 gene has been disrupted. Among otheruses and applications, the animal model developed in this invention areuseful in various screening assays for the identification oftherapeutically useful compounds for the treatment of diseases and/orconditions involving the expression of hnRNP A1 gene.

2. Generation of the Targeting Vector or Nucleic Acid Construct

In order to prepare a knockout animal, a targeting vector or nucleicacid construct containing hnRNP A1 gene is constructed. The nucleic acidconstruct is designed to disrupt the expression of hnRNP A1 gene in acell. The general features of the nucleic acid construct are that itcontains a nucleic acid sequence from one or more regions of the hnRNPA1 gene.

Accordingly, in one aspect, the present disclosure provides a vector ora nucleic acid construct, which includes a genetic engineerheterogeneous nuclear ribonucleoprotein (hnRNP) A1 gene, wherein a firstlocus of recombination sequence 1 is inserted before exon 2 of the hnRNPA1 gene, and recombination sequences 2 (i.e. a first locus and a secondlocus of the recombination sequences 2) flanking a marker gene followedby a second locus of recombination sequence 1 are inserted behind exon 8of the hnRNP A1 gene. The recombination sequence 1 at the first locusundergoes recombination with the recombination sequence 1 at the otherlocus in the presence of its corresponding recombinase. Similarly, therecombination sequence 2 at the first locus undergoes recombination withthe recombination sequence 2 at the other locus in the presence of itscorresponding recombinase. Of note is that, the recombination sequence 1and the recombination sequence 2 belong to different recombinationsequence/recombinase system. In one embodiment, the recombinationsequence 1 and its corresponding recombinase belong to Cre/loxP system,and the recombination sequence 2 and its corresponding recombinasebelong to FLP/frt (FLP recombination target) system. More specifically,the hnRNP A1 gene is disrupted by introduction of Cre recombinase andFLP recombinase.

The Cre/loxP system is a well known system for artificially control geneexpression (Kuhn and Torres, 2002 Methods Mol Biol 180: 175-204). Thesystem begins with the cre gene, which encodes a site-specific DNArecombinase named Cre or cyclic recombinase. A site-specific DNArecombinase means that the Cre protein recombines DNA when it locatesspecific sites in a DNA molecule. These sites are known as loxP or locusof crossover (x) in P1 sequences (SEQ ID NO: 1). When cells that haveloxP sites in their genome also express Cre, the protein may catalyze areciprocal recombination event between the loxP sites, which means thedouble stranded DNA is cut at both loxP sites by the Cre protein andthen ligated back together. As a result, the DNA between the loxP sitesis excised as a circular DNA and subsequently degraded. In order toprepare the nucleic acid construct, the genomic DNA sequence of hnRNP A1gene (SEQ ID NO: 2) is digested with restriction enzymes and the loxPsites are ligated into the genomic DNA sequence at desired locationsusing methods known to those skill artisans or as described in Examplesof this application. The FLP/frt system is functionally analogous to theCre/loxP system. Further examples of recombinases include, but are notlimited to, lambda Int protein, IHF, Xis, Hin, Gin, Cin, Th3 resolvase,TndX and XerD. As to recombination sequences (sites), examples include,but are not limited to, loxP site, frt site (SEQ ID NO: 3), att site,six sites, res sites, rox sites, psi sites and cer site (seeWO2001/1042590).

A marker gene used for selection may be included in the nucleic acidconstruct, so as to identify cells that have been successfullytransfected with the nucleic acid construct of the present invention.The marker gene can be any marker that can be used to detect thepresence of the nucleic acid in a cell. Preferred marker genes areantibiotic resistance genes such as neomycin-resistant gene (Neo, SEQ IDNO:

4), the reporter lacZ gene and the herpes simplex virus thymidine kinasegene (HSV-tk). In one embodiment, the marker sequence is aneomycin-resistant gene. The marker gene may be inserted withrecombination sequences flanking each end of it.

In a preferred embodiment of the present invention, the nucleic acidconstruct is generated by first amplifying sequence homologous to thetarget sequence (such as the hnRNP A1 gene) and then inserting the loxPsequences, and a neomycin resistant gene in combination with frtsequences at each end into the amplified product so that it is flankedby the homologous sequence. Specifically, a loxP sequence is insertedbefore the exon 2 of the endogeneous hnRNP A1 gene, and FLP recombinasetarget (frt) sequences flanking a neomycin-resistant gene followed byanother loxP sequence is inserted behind the exon 8 of the endogenoushnRNP A1 gene. More specifically, the first locus of loxP sequence isinserted between the exon 1 and exon 2 of the endogeneous hnRNP A1 gene,and the neomycin-resistant gene flanked by frt sequences and the secondlocus of loxP sequence are inserted between exon 8 and exon 9.Preferably, the nucleic acid construct is as depicted in FIG. 1.

The nucleic acid construct containing the hnRNP A1 gene, the Cre/loxsystem and the marker gene can be inserted directly into appropriatehost cells such as embryonic stem cells as will be described below or itmay be placed in suitable vectors for amplification prior to insertion.

3. Generation of the Transfected Embryonic Stem Cells

The above nucleic acid constructs or vectors may be transfected into anappropriate host cell using any method known in the art. Varioustechniques may be employed for such purpose, which include and are notlimited to, microinjection of DNA into the nucleus, retrovirus mediatedgene transfer into germ lines, electroporation of embryos,sperm-mediated gene transfer, and calcium phosphate/DNA co-precipitates,transfection or the like.

In a preferred embodiment, the nucleic acid construct is transfectedinto host cells by electroporation. In this process, electrical impulsesof high field strength reversibly permeabilize biomembranes allowing thetransfection of the nucleic acid construct. The pores created duringelectroporation permit the uptake of macromolecules such as DNA into thehost cells.

Any cell type capable of homologous recombination may be used topractice this invention. Preferred cell types include embryonic stem(ES) cells, which are typically obtained from pre-implantation embryoscultured in vitro. The ES cells are cultured and prepared fortransfection of the nucleic acid construct using methods known in therelated art. The ES cells that will be transfected with the targetingvector or nucleic acid construce are derived from embryo or blastocystof the same species as the developing embryo or blastocyst into whichthey are to be introduced. ES cells are typically selected for theirability to integrate into the inner cell mass and contribute to the germline of an individual when introduced into the animal in an embryo atthe blastocyst stage of development. In one embodiment, the ES cells areisolated from the mouse blastocysts, in another embodiment, from the129/SvJ strain.

After transfection into the ES cells, the nucleic acid constructintegrates with the genomic DNA of the cell in order to delet thetranscription of the native hnRNP A1 gene. Preferably, the insertionoccurs by homologous recombination wherein regions of the hnRNP A1 genein the nucleic acid construct hybridize to the homologous hnRNP A1sequence in the ES cell and recombine to incorporate the construct intothe endogenous hnRNP A1 gene.

After transfection, the ES cells are cultured under suitable conditionto detect transfected cells. For example, when the marker gene comprisesan antibiotic resistant marker, the cells are cultured in thatantibiotic. In one embodiment, the neomycin resistant gene is present,and the ES cells are exposed to neomycin analog, G418. ES cells thatexpress the introduced neomycin (Neo) resistant gene are resistant tothe compound G418, whereas ES cells that do not carry the Neo genemarker cannot survive. The DNA and/or protein expression of thesurviving ES cells may be analyzed using Southern Blot technology and/orWestern Blot technology as described in the Examples of thisapplication, in order to identify the ES cells with the properintegration of the construct. The neomycin resistant gene is thenremoved by the introduction of FLP recombinase; and the hnRNP A1 genesegment flanked by the two loxP sequencess, or more specifically theexons 2 to 8 of hnRNP A1 gene, of the survived ES cells may then beremoved by the introduction of Cre recombinase.

4. Generation of the Knockout Animals

The selected ES cells are then injected into a blastocyst of a non-humananimal to form chimeras. The non-human animal may be a mouse, a hamster,a rat or a rabbit. Preferably, the non-human animal is a mouse. Inparticular, the ES cells are inserted into an early embryo usingmicroinjection. For microinjection, 10 to 20 ES cells are collected intoa micropipette and injected into 3 to 5 day old bastocysts recoveredfrom female mice. The injected blasocysts are re-implanted into a fostermother. When the pups are born, typically 18 to 20 days later, they arescreened for the presence of nucleic acid construct of this invention.In one embodiment, DNA collected from the tail tissues of the pups maybe screened using Southern Blot and/or PCR technique as described inExamples of this disclosure. The heterozygotes are identified and arethen crossed with each other to generate homologous knockout animals.

Accordingly, the present invention provides a transgenic non-humananimal and its progeny, whose genome comprising a disruption in hnRNP A1gene, wherein the animal has a decreased (i.e., heterozygous disruption)or null (i.e., homozygous disruption) expression level of the hnRNP A1gene as compared to that of a wild-type animal. The present inventionalso provides cells or tissues, including immortalized cell lines andprimary cells or tissues, derived from the transgenic non-human animaland its progeny. The expression of the hnRNP A1 gene may be partially orcompletely disrupted. In the case when a complete disruption occurs, thelevel of hnRNP A1 gene is not detectable by Southern blotting. In oneembodiment, the disruption affects at least two exons within the hnRNPA1 gene. In another embodiment, the exons are exons 2 to 8.

In one embodiment, the transgenic non-human animal and its progeny aremice, hamsters, rats or rabbits. In another embodiment, the transgenicnon-human animal and its progeny are mice. The homozygous disruption inthe hnRNP A1 gene of the transgenic mouse results in damaged function ofexonic RNA splicing, a reduced weight relative to a wild-type controlmouse at embryonic stage, and/or perinatal mortality. Furthermore, thetransgenic mouse with heterozygous disruption in the hnRNP A1 gene ispredisposed to premature aging diseases and/or virus infective diseases.

The present invention further provides a method of preparing transgenicnon-human animal with decreased or null expression level of hnRNP A1gene comprising steps of inserting into the genome of the embryonic stemcell derived from the non-human animal the nucleic acid construct of thepresent invention, injecting the embryonic stem cell into a blastocystof the non-human animal after introduction of appropriate recombinases,and implanting the blastocyst into the uterus of a foster mother of thenon-human animal. In one embodiment, the non-human animal is a mouse, ahamster, a rat or a rabbit. In another embodiment, the non-human animalis a mouse.

In a preferred embodiment, the method includes steps of: (1) obtaining anucleic acid sequence containing a hnRNP A1 gene or a portion thereof;(2) preparing a nucleic acid construct of the present invention; (3)transfecting the nucleic acid construct into an ES cell; (4) selectingan ES cells that has integrated the nucleic acid construct into itsgenome; (5) introducing FLP and Cre recombinases to the selected EScells in step (4) to remove the marker gene and exons 2 to 8 of thehnRNP A1 gene and generate a deleted hnRNP A1 gene; (6) introducing theselected ES cell in step (5) into a blastocyst to form a chimerablastocyst; (7) implanting the chimeric blastocyst into a pseudopregnantmother, wherein the mother gives birth to a chimeric animal having thedisrupted hnRNP A1 gene in its genome; (8) crossing the chimeric animalobtained in step (7) with a normal animal to obtain a heterozygousknockout animal; (9) repeating the crossing defined in step (8) at least1 time to generate another heterozygous knockout animal; and (10)crossing the heterozygous knockout animals obtained in step (8) and (9)with each other to generate a homozygous or heterozygous hnRNP A1knockout animal. Preferably, the crossing step defined in step (8) isrepeated at least 2, 3, 4 or 5 times to generate the heterozygousknockout animals. More preferably, the crossing step defined in step (8)is repeated at least 5 times to generate the heterozygous knockoutanimals.

In a preferred embodiment, the non-human heterozygous knockout animal isa heterozygous hnRNP A1 knockout mouse.

5. Use of the Knockout Animals

The knockout animals of the present invention are useful for studyingthe function of hnRNP A1 gene and diseases wherein the hnRNP A1 gene isimplicated, including neurodegenerative disease and cancer. Hence, thenon-human hnRNP A1 knockout animals of the present disclosure are usefulfor identifying therapeutic compounds that may be useful in preventingand/or treating any of these diseases.

Accordingly, the present disclosure provides a method of screening acompound for potential use in prevention and/or treatment ofneurodegenerative disease or cancer. The method includes steps ofrespectively administering a test compound to a non-human animalcomprises a disruption in hnRNP A1 gene or primary cells or tissuesderived therefrom and a wild-type non-human animal or primary cells ortissues derived therefrom; and assessing functions of exonic RNAsplicing in each of the non-human animal, cell, or tissue defined above,prior to and after a given time period of the administration; andcomparing the assessment results to determine effectiveness of the testcompound.

In one embodiment, the non-human animal is a mouse, a hamster, a rat ora rabbit. In another embodiment, the non-human animal is a mouse.

In a further embodiment, the primary cells or tissues derived from thehnRNP A1 gene-disrupted mouse are prepared by a method well known in theart (Kazutoshi et al., Nature Protocols (2007), 2: 3081-3089; and Yen etal., Environmental Health Perspectives (2010), 118: 949-956). Theprimary cells can be fibroblasts from the non-human animal embryos ormyoblasts from the neonatal non-human animal. The the non-human animalmay be, but not limited to, mouse. Briefly, the process for preparingmouse embryonic fibroblasts (MEF) includes the following steps: (1)isolating mouse embryos at day 13.5 and removing the head, visceraltissues and gonads from the isolated embryos; (2) hashing out theremaining embryonic body and incubating in a solution containing trypsinand EDTA under 37° C.; (3) dissociating the embryonic body inappropriate medium to form a cell suspension; (4) centrifuging the cellsuspension to enrich the MEFs; and (5) culturing the MEFs in appropriatemedium with suitable cellular concentration. The primary myoblasts ofcan be prepared from the forelimb and hind limb of neonatal mousethrough the following steps: (1) removing surrounding connective tissueand mincing the muscles into small pieces; (2) digesting the mincedmuscles with collagenase; (3) incubating the digested muscles withtrypsin to dissociate cells; and (4) collecting the dissociated cells bycentrifugation and incubating the cells in appropriate medium.

The following examples are provided to illustrate the present inventionwithout, however, limiting the same thereto.

EXAMPLES

The process for generating the hnRNP A1 knockout mice and uses thereofof the present disclosure will be illustrated in further detail withreference to several examples below, which are not intended to limit thescope of the present disclosure.

Example 1 The Generation of hnRNP A1 Knockout Mice

1.1 Constructing the Target Vector

The hnRNP A1 targeting vector was generated by deleting exons 2 to 8 ofthe hnRNP A1 gene. This targeting construct was created usingrecombineering techniques in a 129S7/AB2.2 bacterial artificialchromosome containing the hnRNP A1 gene (clone bMQ-281N24, Geneservice,Cambridge, UK). A loxP site was inserted before the exon 2 of hnRNP A1.More particularly, the loxP site was inserted between exon 1 and exon 2of hnRNP A1 gene. A neomycin resistance gene was inserted into the hnRNPA1 gene for the enhancement of selecting positively targeted embryonicstem cells (ES) clones in the presence of neomycin analog G-418. A frtsites flanking the neomycin resistance cassette followed by another loxPsite was then inserted behind exon 8. Specifically, “behind exon 8”means between exon 8 and exon 9.

FIG. 1 is a schematic drawing illustrating the constructed vector ofExample 1.1 having DNA fragments with exons 2 to 8 of hnRNP A1 beingdeleted for generating hnRNP A1 knockout mice.

1.2 Selection of Targeted Embryonic Stem (ES) Cell Clones

In order to incorporated the mutated hnRNP A1 gene in the targetingvector into the ES cells for targeting homologous recombination tooccur, the targeting construct of Example 1.1 was electroporated into129 ES cells. The transfected ES cells were then selected by neomycinanalog G418. In principle, those ES cells that were targeted and thuscarried the mutated hnRNP A1 alleles could survive in the cultureenvironment containing G148 because of the presence of a built-inneomycin resistant gene in the mutated vector. The non-targeted wildtype ES cells would die because of the lack of the neomycin gene in thepresence of G418.

The surviving targeted ES cells clones were microscopically picked andcultured separately. The individually grown ES cell clones wereharvested and isolated for genomic DNAs. The extracted DNAs wereanalysed on their restriction fragment patterns using genomic Southernblot technology to select for the replacement targeting throughhomologous recombination and to differentiate it from the unwanted geneinsertion events. The neomycin resistance cassette was deleted byintroduced Flp recombinase; and the gene segment flanked by the two loxPsites, containing exons from 2 to 8 of hnRNP A1, of the targeted EScells was deleted by introduced Cre recombinase. The clones showed theband pattern of having 2 site were hnRNP A1 targeted +/− heterozygousknockouts. They were selected for blastocyst injection after expansion.

1.3 Generation of Mouse hnRNP A1 Knockout Line

The targeted ES cell clones of example 1.2 were expanded according tostandard procedures. The ES cells were then microinjected intoblastocysts recovered from female C57BL/6 mice. The injected blastocystswere re-implanted to female BALB/C mice as foster mothers for theembryos. Approximately 30 to 40 blastocysts were implanted to eachfoster mother. The foster mothers were maintained in sterile conditions.Litters were born 18-20 days later. Among the newly born pups there were3 male and 2 female chimeras with different degree of agouti color furs.To determine whether these chimeras had targeted hnRNP A1 ES cellsdeveloped into germ cells, the chimeras were mated with the wild-typeC57BL/6 mice to generate the F1 mice. The F1's were screened andgenotyped for germline transmission.

PCR methods were used for genotyping the tail DNAs of the mice. Theprimers, A1U (5′-tatagcgggatgtgacgtgttttg-3′, SEQ ID NO: 5) and WT-L(5′-aatgaatcaacaccccgcaacaac-3′, SEQ ID NO: 6), were used to show thepresence of the wild type allele. The deleted allele was detected by theprimers A1U and KO-L (5′-actgcacccacaatgctttaagag-3′, SEQ ID NO: 7).Result is depicted in FIG. 2A.

The F1 mice were also analyzed for their expression of hnRNP A1 proteinusing western blot analysis. Briefly, mouse tissues such as heart andbrain, were collected and homogenized in RIPA buffer (50 mM Tris-HCl,pH8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1% SDS,1× protease inhibitor cocktails and 1 mM PMSF) at 4° C. for 20 min. Thelysates were subjected to SDS-PAGE and immunoblotting. The blots wereprobed with anti-hnRNP A1 antibody (Sigma, Saint Louis, Mo., USA)) oranti-beta-actin antibody (Santa Cruz, Santa Cruz, Calif., USA) anddetected by ECL chemiluminescence kit (GE Healthcare, Piscataway, N.J.,USA). Result is depicted in FIG. 2B.

The mice were bred in a specific pathogen-free facility and treatedaccording to the Guide for the Care and Use of Laboratory Animals issuedby National Research Council (Taiwan, Republic of China). Embryos of thehnRNP A1 heterozygotes, which were formally named as B6.129-Hnrnpa1^(tm1Cfy) , had been deposited in Rodent Model ResourceCenter of National Laboratory Animal Center (RMRC-NLAC) in Taiwan withthe deposit No. RMRC13102. The hnRNP A1 heterozygotes carrying onedeleted allele were interbred to achieve the homozygous hnRNP A1 nullmice.

Example 2 Characterization of the hnRNP A1 Knockout Mice

2.1 hnRNP A1^(−/−) Mice Are Embryonic Lethality Or Dead After Birth

After heterozygous intercross as described in Example 1.3, 18 hnRNPA1^(+/+), hnRNP A1^(+/−) and 8 hnRNP A1^(−/−) pups were born among 10litters. The heterozygous mice appeared completely normal and fertile.However, the hnRNP A1^(−/−) pups showed small body size and none ofhomozygous mutant mice was alive after birth. In addition, the number ofthe homozygous mutant mice was lower than the predicted number ofMendel's Law of Inheritance. These results suggest that a mouse lack ofhnRNP A1 results in embryonic lethality or immediately dead after birth.

In order to determine the embryonic lethality, we examined the genotypeof embryos at E18.5 from heterozygous intercross. There were 20 hnRNPA1^(+/+), 36 hnRNP A1^(+/−) and 16 hnRNP A1^(−/−) embryos at E18.5 among10 litters. The p value from chi-square analysis is 0.8007. The p value,greater than 0.05, means the genotype of the embryos following Mendel'sLaw of Inheritance of the ratio 1:2:1. In addition, the body length ofhnRNP A1^(−/−) embryos range from 1.0 to 2.1 cm, lower than the averagebody length of E18.5 (2.2-2.5 cm). This data indicates that thedevelopment of hnRNP A1^(−/−) embryos ceased at different developmentalages. Overall, the results support the hypothesis that a mouse lack ofhnRNP A1 results in embryonic lethality or immediately dead after birth.

2.2 hnRNP A1^(−/−) Mice Display Embryonic Growth Retardation

In this example, morphological and histological properties of the hnRNPA1^(−/−) mice at E18.5 were examined. The hnRNP A1^(−/−) embryodisplayed growth retardation (FIG. 3A). The internal organs of the hnRNPA1^(−/−) embryo did not appear obviously abnormal (FIG. 3B).Histological analysis were then performed on the embryos. The sectionswere stained with hematoxylin and eosin (HE). The hnRNP A1^(−/−) micedisplay diaphragm and urinary bladder defects at E18.5. Histologicalanalysis showed that the diaphragm of hnRNP A1^(−/−) mouse displayedsarcoplasmic degeneration and fibrous tissues infiltration (FIG. 4B)compared with hnRNP A1^(+/+) embryo (FIG. 4A). Moreover, the urinarybladder of hnRNP A1^(−/−) mouse showed hyperplasia of transitional cellsand appeared several degenerative cells (FIG. 4D) compared with hnRNPA1^(+/+) embryo (FIG. 4C). These results suggest that a mouse lack ofhnRNP A1 results in developmental retardation, which may be multi-organsaffected.

2.3 Heterozygous hnRNP A1^(+/−) Adult Mice Express Low hnRNP A1 Proteinin Organs

Since the gene number of hnRNP A1 in heterozygous hnRNP A1^(+/−) adultmice are lower than homozygous hnRNP A1^(+/+) mice. The protein levelsof hnRNP A1 in the organs of these mice were determined. The organs,such lung, testis and brain, from these mice were collected and usingWestern blot to detect the amount of hnRNP A1 protein. The resultsshowed that heterozygous hnRNP A1^(+/−) adult mice expressed lower hnRNPA1 protein than hnRNP A1^(+/+) mice in these organs (FIG. 5). Theseresult results demonstrate that even with the normal phenotype, theheterozygous hnRNP A1^(+/−) adult mice express low hnRNP A1 protein insome organs.

It will be understood that the above description of embodiments is givenby way of example only and that various modifications may be made bythose with ordinary skill in the art. The above specification, examplesand data provide a complete description of the structure and use ofexemplary embodiments of the invention. Although various embodiments ofthe invention have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those with ordinary skill in the art could make numerous alterations tothe disclosed embodiments without departing from the spirit or scope ofthis invention.

All publication, patents and patent application are herein incorporatedby reference in their entirety to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

1. A nucleic acid construct comprising a genetic engineered heterogeneous nuclear ribonucleoprotein (hnRNP) A1 gene, wherein a first locus of recombination sequence 1 is inserted before exon 2 of the hnRNP A1 gene, and recombination sequences 2 flanking a marker gene followed by a second locus of recombination sequence 1 are inserted behind exon 8 of the hnRNP A1 gene; wherein the recombination sequence 1 and recombination sequence 2 undergo recombination with the same recombination sequence at the other locus in the presence of their corresponding recombinase.
 2. The nucleic acid construct of claim 1, wherein the marker gene detects the presence of the nucleic acid construct in a cell.
 3. The nucleic acid construct of claim 1, wherein the marker gene is an antibiotic resistance gene, a reporter lacZ gene, or a herpes simplex virus thymidine kinase gene (HSV-tk).
 4. The nucleic acid construct of claim 3, wherein the antibiotic resistance gene is neomycin-resistant gene.
 5. The nucleic acid construct of claim 1, wherein the recombination sequence 1 is a crossover (x) in P1 (loxP) site and the recombination sequences 2 is a FLP recombinase target (frt) site.
 6. The nucleic acid construct of claim 5, wherein the hnRNP A1 gene is disrupted by introduction of Cre recombinase and FLP recombinase.
 7. A transgenic mouse whose genome comprising a homozygous disruption in hnRNP A1 gene, wherein the disruption results in the transgenic mouse having a damaged function of exonic RNA splicing, a reduced weight relative to a wild-type control mouse at embryonic stage, and perinatal mortality.
 8. The transgenic mouse of claim 7, wherein the disruption affects at least two exons within the hnRNP A1 gene.
 9. The transgenic mouse of claim 8, wherein the exons are exons 2 to
 8. 10. A transgenic mouse whose genome comprising a heterozygous disruption in hnRNP A1 gene.
 11. The transgenic mouse of claim 10, wherein the disruption affects at least two exons within the hnRNP A1 gene.
 12. The transgenic mouse of claim 11, wherein the exons are exons 2 to
 8. 13. The transgenic mouse of claim 10, wherein the transgenic mouse is predisposed to premature aging diseases and/or virus infective diseases.
 14. A method of screening a compound for potential use in prevention and/or treatment of neurodegenerative disease or cancer, which comprises, (a) administering a test compound to the transgenic mouse of claim 7 or primary cells or tissues derived therefrom; (b) administering a test compound to a wild-type mouse or primary cells or tissues derived therefrom; (c) assessing functions of exonic RNA splicing in each of the mice, cell, or tissues from step (a) and (b), prior to and after a given time period of the administration; and (d) comparing the assessment results of step (c) to determine effectiveness of the test compound. 